The present invention relates generally to the non-enzymatic glycosylation of amyloid proteins and the often consequent formation of advanced glycosylation endproducts (AGEs). Formation of AGE-amyloid can result in disease conditions or complications. The invention particularly relates to compositions and methods for the prevention and treatment of amyloidosis associated with neurodegenerative diseases, in particular Alzheimer""s disease, and amyloidosis associated with Type II (adult onset) diabetes.
Amyloidosis generally refers to a physiological condition which involves deposition of insoluble polypeptides, termed amyloid polypeptides or amyloid proteins. There are a wide range of amyloid proteins found in various tissues throughout a subject, and a number of pathological conditions associated with various amyloidoses. For example, multiple myeloma can result in amyloidosis with the immunoglobulin proteins. Idiopathic familial Mediterranean fever also involves systemic amyloidosis. Perhaps the best known disease associated with amyloidosis is Alzheimer""s disease.
Alzheimer""s disease (AD) affects more than 30% of humans over 80 years of age, and as such, represents one of the most important health problems of developed countries (Evans et al., 1989, JAMA 262:2551-56; Katzman and Saitoh, 1991, FASEB J. 280:278-286). The etiology and pathogenesis of this progressive dementia is poorly understood, but symptomatic disease is associated with deposits of amyloid plaques, cerebrovascular amyloid and neurofibrillary tangles in the brain and cerebrovasculature. The number of plaques in AD patients"" brains are typically 5- to 10 fold greater than in age-matched healthy controls. Increased levels of plaques may result from increased rate of synthesis of the components of the plaques, decreased rate of degradation, or some combination of the two.
The primary protein component of plaques is the 42 amino acid (4.2 kDa) beta-Amyloid Peptide (xcex2AP), which derives from a family of larger Amyloid Peptide Precursor (APP) proteins (Glenner and Wong, 1984, Biochem. Biophys. Res. Commun. 120:885-890; Glenner and Wong, 1984, Biochem. Biophys. Res. Commun. 122:1131-35; Goldgaber et al., 1987, Science 235:8778-8780; Kang et al., 1987, Nature 325:733-736; Robakis et al., 1987, Proc. Natl. Acad. Sci. USA 84:4190-4194; Tanzi et al., 1987, Science 235:880-884). The process of amyloidosis is poorly understood, but requires at least xcex2AP. Recent evidence shows that xcex2AP may be found in extracellular spaces like cerebrospinal fluid (CSF) of the brain and conditioned media of many cell types. Since increased amountand of amyloid deposits are present in AD brains, one simple hypothesis is that increased xcex2AP production leads to increased amyloidosis. Messenger RNAs encoding the APP precursors of xcex2AP increase about 2-fold in AD brains, which has suggested to some a possible 2-fold increase in rates of translation, which may explain increased amyloid plaque formation (e.g., Jacobsen et al., 1991, Neurobiol. Aging 12:585-592, and references cited therein; Palmert et al., 1989, Prog. Clin. Biol. Res. 317:971-984; Tanaka et al., 1990, Rinsho Byori 38:489-493; Tanaka et al., 1989, Biochem. Biophys. Res. Commun. 165:1406-1414). An example of an increased efficiency of xcex2AP production that correlates with increased plaque levels is found in a rare genetically linked familial form of Alzheimer""s disease (Cai et al., 1992, Science 259:514-516; Citron et al., 1992, Nature 360:672-674; Mullan et al., 1992, Nature Genet. 1:345-347), known as a Swedish disease involving a double lysine-methionine (KM) to asparagine-leucine (NL) mutation in APP near the amino-terminus of xcex2AP. This mutation increases the release of extracellular xcex2AP in cultured cells. However, while this observation may partly explain amyloidosis in the Swedish disease (and Down""s Syndrome), xcex2AP peptide levels in CSF of AD and healthy patients are the same (Oosawa et al., 1993, Soc. Neurosci. Abst. 19:1038; Shoji et al., 1992, Science 258:126-129). Thus, although healthy subjects appear to possess similar quantities of xcex2AP as AD patients, they nevertheless fail to accumulate the high number and amount of amyloid plaques seen in their AD counterparts.
Post-translational events may contribute to amyloidosis. Beyond increased rates of translation, physiological events such as greater efficiency of xcex2AP production from its precursor, aggregation into fibrillar structures, and resistance to proteolysis may unbalance degradative processes, resulting in plaque formation.
Aggregation of the components of amyloid is a critical step in the development of amyloidosis. Once formed, fibrillar aggregates of xcex2AP are extremely stable and not easily degraded. Amyloid plaques may be purified by their resistance to solubilization in boiling SDS and digestion with a variety of proteases. Additional treatment with 80% formic acid or 6M guanidine thiocyanate eventually solubilizes a portion of the plaque material. The solubilized protein is primarily the 42 amino acid xcex2AP. Yet even after these harsh denaturation treatments, dimers, tetramers and large molecular weight aggregates containing immunoreactive xcex2AP are found. This resistance to solubilization into soluble or monomeric components suggests extensive protein modifications.
Further experiments have shown that primary neuronal cultures treated with full length xcex2AP 1-42 in soluble form remain viable. Thus, soluble xcex2AP 1-42 shows no toxicity. In contrast, cultures treated with insoluble aggregates of xcex2AP 1-42 show a toxic response (Pike et al., 1991, Eur. J. Pharm. 207:367-368; Pike et al., 1993, J. Neurosci. 13:1676-87). This experiment suggests that the toxicity of xcex2AP is related to its state of aggregation. Thus, an understanding of the mechanism forming fibrils and/or insoluble aggregates from soluble xcex2AP may be critical to preventing toxicity and resulting neurodegenerative disease.
In the absence of increased soluble xcex2AP in most cases of AD, the question remains how amyloid accumulates to a greater degree at different rates. Synthetic xcex2APs corresponding to the first 28, 40, or 42 amino acids of xcex2AP (i.e., xcex2AP 1-28, xcex2AP 1-40 and xcex2AP 142, respectively) display concentration-dependent aggregation kinetics in in vitro incubations. Fibrillar aggregates form in vitro and these appear similar to brain xcex2-amyloid fibrils at the morphological level using electron microscopy and at the light microscopy and spectroscopic levels using Congo Red and Thioflavin stains.
The more rapid kinetics of aggregation observed at xcexcM concentrations of soluble xcex2AP in vitro are only of limited relevance for insight into the mechanism of fibril formation in vivo. At lower xcex2AP concentrations, for instance in the physiological range of about 5 nM, there is a considerable lag period before measurable aggregate is formed in vitro. This observation suggests that the rate limiting step in aggregation could be formation of a xe2x80x9cnucleusxe2x80x9d or xe2x80x9cseedxe2x80x9d upon which additional xcex2AP can rapidly accumulate.
There are two broad types of diabetes: Type I (childhood onset diabetes), which is associated with destruction of the pancreatic beta cells and loss of insulin, and other hormones, produced by these cells, and is treated with insulin; and Type II (adult onset diabetes), which is associated with insulin resistance. Type II diabetics can be further divided into Type IIA, characterized by high blood pressure, obesity and insulin resistance, and Type IIB, which includes lean individuals, obese insulin sensitive individuals, and young individuals. Perhaps the most significant distinction between Type I and Type II diabetes is the absence of autoimmune disease in Type II diabetes; otherwise, this syndrome is characterized by a similarly diverse array of symptoms and causes.
One common characteristic of Type II diabetics is the presence of amyloid plaques in the pancreas. Such plaques are found in 90% of Type II diabetics upon autopsy. As with Alzheimer""s disease, the presence of amyloid plaques in the affected organ cannot be conclusively demonstrated until autopsy (see, Edgington, 1994, Bio/Technology 12:591). Two groups independently identified the major component of pancreatic amyloid plaques as a 37 amino acid polypeptide termed islet amyloid polypeptide (IAPP) (Westermark et al., 1987, Proc. Natl. Acad. Sci. USA 84:3881-85; Westermark et al., 1987, Am. J. Physiol. 127:414-417), or amylin (Cooper et al., 1987, Proc. Natl. Acad. Sci. USA 84:8628-32; Cooper et al., 1988, Proc. Natl. Acad. Sci. USA 85:7763-66); the peptides identified by both groups appear to be interchangeable (Amiel, 1993, Lancet 341:1249-50). In its soluble form, amylin antagonizes insulin, and thus appears to have a role in the regulation of bloodstream glucose levels (see, Edgington, supra).
However, at high concentration, amylin, like xcex2AP, aggregates in a xcex2-pleated sheet structure, and forms fibrils that appear to be toxic (Lorenzo et al., 1994, Nature 368:756-760). In particular, this paper reports that human amylin fibrils are toxic to insulin-producing xcex2-cells of the adult pancreas of rats and humans. Amylin fibrils appear to induce islet cell apoptosis, leading to cell dysfunction and death in Type II diabetes mellitus (Lorenzo et al., supra).
The reaction between glucose and proteins has been known for some time. Its earliest manifestation was in the appearance of brown pigments during the cooking of food. In 1912, Maillard observed that glucose or other reducing sugars react with amino acids to form adducts that undergo a series of dehydrations and rearrangements to form stable brown pigments (Maillard, 1912, C.R. Acad. Sci. 154:66-68).
In the years that followed the initial discovery by Maillard, food chemists studied the hypothesized reaction in detail and determined that stored and heat-treated foods undergo nonenzymatic browning as a result of the reaction between glucose and polypeptide chains, and that the proteins thereby become crosslinked and exhibit decreased.bio-availability. At this point, it was determined that the pigments responsible for the development of the brown color as a result of protein glycosylation possessed characteristic spectra and fluorescent properties; however, the chemical structure of the pigments had not been specifically elucidated.
The reaction between reducing sugars and food proteins discussed above was found in recent years to have its parallel in vivo. Thus, the nonenzymatic reaction between glucose and the free amino groups on proteins to form a stable amino, 1-deoxy ketosyl adduct, known as the Amadori product, has been shown to occur with hemoglobin, wherein a rearrangement of the amino terminal of the B-chain of hemoglobin by reaction with glucose forms the adduct known as hemoglobin A1c. The reaction has also been found to occur with a variety of other body proteins, such as lens crystallin, collagen and nerve proteins (see Bunn et al., 1975, Biochem. Biophys. Res. Commun. 67:103-109; Koenig et al., 1975, J. Biol. Chem. 252:2992-2997; Monnier and Cerami, in Maillard Reaction in Food and Nutrition, ed. Waller, G. A., American Chemical Society 1983, pp. 431-448; and Monnier and Cerami, 1982, Clinics in Endocrinology and Metabolism 11:431-452).
Moreover, brown pigments with spectral and fluorescent properties similar to those of late-stage Maillard products have also been observed in vivo in association with several long-lived proteins, such as lens proteins and collagen from aged individuals. An age-related linear increase in pigment was observed in human dura collagen between the ages of 20 to 90 years (see Monnier and Cerami, 1981, Science 211:491-493; Monnier and Cerami, 1983, Biochem. Biophys. Acta 760:97-103; and Monnier et al., 1984, xe2x80x9cAccelerated Age-Related Browning of Human Collagen in Diabetes Mellitusxe2x80x9d, Proc. Natl. Acad. Sci. USA 81:583-587). Interestingly, the aging of collagen can be mimicked in vitro in a much shorter period of time by crosslinking induced by incubation in solution with relatively high concentrations of glucose. The capture of other proteins and the formation of adducts by collagen, also noted, is theorized to occur by a crosslinking reaction, and is believed to. account, for instance, for the observed accumulation of albumin and antibodies in kidney basement membrane (see Brownlee et al., 1983, J. Exp. Med. 158:1739-1744; and Kohn et al., 1984, Diabetes 33:57-59).
Glucose and other reducing sugars attach non-enzymatically to the amino groups of proteins in a concentration-dependent manner. Over time, these initial Amadori adducts can undergo further rearrangements, dehydrations and cross-linking with other proteins to accumulate as a complex family of structures referred to as Advanced Glycosylation Endproducts (AGEs). Substantial progress has been made toward the elucidation of the role and clinical significance of advanced glycosylation endproducts, so that it is now acknowledged that many of the conditions heretofore attributed to the aging process or to the pathological effects of diseases such as diabetes, are attributable at least in part to the formation of AGEs in vivo.
As noted above, advanced glycosylation end products tend to accumulate on molecules with long half-lives, especially under conditions of relatively high sugar concentration. Thus, AGE accumulation can be indicative of protein half-life, sugar concentration, or both. These factors have important consequences. Numerous studies have suggested that AGEs play an important role in the structural and functional alteration which occurs during aging and in chronic disease. Additionally, advanced glycosylation endproducts are noted to accumulate to a greater extent in diabetic and other diseased tissue than in normal tissue.
The xe2x80x9cfamilyxe2x80x9d of AGEs includes species which can be isolated and characterized by chemical structure, some being quite stable, while others are unstable or reactive. The reaction between reducing sugars and the reactive groups of proteins may initiate the advanced glycosylation process. This process typically begins with a reversible reaction between the reducing sugar and the reactive group to form a Schiff base, which proceeds to form a covalently-bonded Amadori rearrangement product. Once formed, the Amadori product undergoes further rearrangement to produce the AGE-modified compound.
In U.S. Pat. No. 4,665,192, a fluorescent chromophore was isolated and identified that was found to be present in certain browned polypeptides, such as bovine serum albumin and poly-L-lysine, and was assigned the structure 2-(2-furoyl)-4(5)-2(furanyl)-1H-imidazole. More recently, other advanced glycosylation products have been identified, e.g., as described in Farmar et al., U.S. Pat. No. 5,140,048, issued Aug. 18, 1992; pyrraline (Hayase et al., 1989, xe2x80x9cAging of Proteins: Immunological Detection of a Glucose-derived Pyrrole Formed during Maillard Reaction in Vivoxe2x80x9d, J. Biol. Chem. 263:3758-3764); and pentosidine (Sell and Monnier, 1989, xe2x80x9cStructure Elucidation of a Senescence Cross-link from Human Extracellular Matrixxe2x80x9d, J. Biol. Chem. 264:21597-602).
Based on their knowledge of the role of AGEs in disease, the present inventors have sought to identify factors that enhance aggregation of xcex2AP, and more importantly to identify agents and methods to inhibit the action of such factors and thus prevent amyloidosis, e.g., in Alzheimer""s disease and other amyloid diseases. More particularly, the invention seeks to discover the relationship between advanced glycosylation endproduct formation and amyloidosis. Prior to the instant invention, there has been no appreciation of a relationship between amyloidosis and advance glycosylation endproduct formation.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
The present invention is broadly directed to the discoveries about the nature of AGE modification of amyloidogenic polypeptides, and the consequences of such modification for the pathology and therapeutic treatment of diseases or disorders associated with amyloidosis.
In particular, the inventors have discovered that AGE-amyloid polypeptides, in particular AGE-xcex2 amyloid peptide (xcex2AP), facilitate further aggregation of amyloid polypeptides, whether such amyloid polypeptides are AGE-modified or not.
The inventors have further related this discovery to the enhanced ability of AGE-amylin polypeptides to facilitate aggregation of amylin (whether AGE-modified or not), resulting in amyloidosis of pancreatic tissue and death of pancreatic islet cells.
Thus, the invention relates to a method of modulating AGE-amyloid polypeptide-mediated amyloidosis in a mammal by controlling the formation of AGE-amyloid polypeptides. In a specific aspect of the invention, aggregation of xcex2AP and amylin have been determined to be enhanced by the glycosylation reaction of xcex2AP or amylin to form AGE-xcex2AP or AGE-amylin as defined herein. Accordingly, the invention particularly extends to a method for modulating the in vivo aggregation of xcex2AP and associated neurodegenerative amyloidosis by controlling the formation and presence of AGE-xcex2AP. The invention further particularly extends to a method for modulating the in vivo aggregation of amylin and associated pancreatic islet cell amyloidosis by controlling formation and presence of AGE-amylin.
It has also been discovered that individuals suffering from an amyloidogenic disease have more AGEs associated with the amyloid polypeptides that form the amyloid plaques characteristic of the disease. The presence and level of AGE-amyloid polypeptides may reflect the total body burden of amyloid polypeptides and their age. In particular, patients with Alzheimer""s disease have more AGEs associated with xcex2AP than normal individuals of the same age, and patients with Type II diabetes may have more AGEs associated with amylin than normal individuals. Since the absolute levels of xcex2AP in AD and normal individuals is about the same, the presence of AGE-xcex2AP can be indicative or predictive of AD.
A corresponding diagnostic utility comprises the measurement of the course and extent of amyloidosis by a measurement of the presence and amount of AGE-amyloid polypeptides, and particularly AGE-xcex2AP and AGE-amylin, as defined herein. An assay is included that may use the AGE-amyloid polypeptide of the present invention to identify disease states characterized by the presence of the AGE-amyloid polypeptide. Additionally, such an assay can be utilized to monitor therapy and thus adjust a dosage regimen for a given disease state characterized by the presence of the AGE-amyloid polypeptide. In specific embodiments, the diagnostic assays of the invention may be used to monitor the presence or level of AGE-xcex2AP or AGE-amylin.
As noted above, AGE-amyloid polypeptide is useful as a marker of a variety of conditions in which the fluctuation in amyloid polypeptide levels may reflect the presence or onset of dysfunction or pathology. Moreover, AGE-amyloid polypeptide is useful alone and in conjunction with known carriers and delivery vehicles; such as liposomes, for the transport of therapeutic and other agents, including in certain instances the AGE moieties themselves, across membranes and epithelial layers, for example, and particularly the blood brain barrier, to particular sites in a patient for treatment. The particular site of interest may be an amyloid plaque that recognizes an AGE-amyloid polypeptide, such as the AGE-xcex2AP, or AGE-amylin, or a portion thereof.
The presence of high levels of AGE-amyloid polypeptides in amyloidogenic diseases indicates that the normal clearance mechanisms for such polypeptides are faulty. Therefore, in a further aspect, the present invention provides compositions and methods for stimulating or inducing mechanisms of recognition and removal of AGE-amyloid in an animal, i.e., the invention contemplates activation of the scavenger system in an animal""s body to remove the amyloid plaques. Such scavenger systems include the activity of phagocytic cells, e.g., macrophages and, in neural tissue, microglial cells.
Accordingly, the invention provides for stimulating or activating the natural scavenger systems by administration of stimulatory agents, including but not limited to, an advanced glycosylation endproduct, an AGE bound to a carrier, the fluorescent chromophore 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) bound to a carrier, a monokine (e.g., lymphokine or cytokine) that stimulates phagocytic cells in the animal to increase the activity of recognizing and removing AGE-amyloid, and mixtures thereof. In a specific aspect, the AGE is an AGE-amyloid polypeptide.
Accordingly, the invention provides a method of preparing AGE-amyloid polypeptide, in particular AGE-xcex2AP or AGE-amylin, which comprises incubation with an advanced glycosylation endproduct or a compound which forms advanced glycosylation endproducts for a length of time sufficient to form said AGE-amyloid polypeptide, e.g., AGE-xcex2AP or AGE-amylin.
Pharmaceutical compositions are also disclosed that comprise an AGE-amyloid polypeptide in combination with a pharmaceutically acceptable carrier. Such pharmaceutical compositions may include an additional active agent(s) in some instances, and may be prepared and used for oral, parenteral or topical, e.g., transdermal, sublingual, buccal or transmucosal delivery. As stated, the pharmaceutical compositions can be in the form of a liposome in certain instances.
Generally, the therapeutic methods of the present invention contemplate the inhibition of in vivo amyloid aggregation by the administration of an agent or a pharmaceutical composition containing such agent or a plurality of such agents, for the inhibition of the formation of advanced glycosylation endproducts involving any or all of the amyloid polypeptide and amyloid precursor polypeptide, and materials subject to such in vivo aggregation. Such agents comprise antagonists of advanced glycosylation, and include antibodies to AGEs, antibodies to AGE-amyloid polypeptide, in particular AGE-xcex2AP and AGE-amylin, as well as other ligands that would bind and neutralize the foregoing antigens. Suitable agents may also be selected from those agents that are reactive with an active carbonyl moiety on an early glycosylation product, and preferably are selected from aminoguanidine, a-hydrazinohistidine, analogs of aminoguanidine, and pharmaceutical compositions containing any of the foregoing, all as recited in detail herein. The invention set forth herein contemplates the discovery of additional agents that may then be used in like fashion and for like purpose.
Accordingly, it is a principal object of the present invention to modulate and control the in vivo aggregation of amyloid polypeptides leading to amyloidosis by controlling the formation of advanced glycosylation endproducts (AGEs), and particularly AGEs involving such amyloid polypeptides.
It is a further object of the present invention to provide a method for the prognosis, monitoring, and/or diagnosis of conditions in which abnormal amyloid accumulation is a characteristic, by detecting and measuring the presence and extent of AGE-amyloid polypeptide formation.
It is a still further object of the present invention to provide a method for diagnosing and treating diseases associated with amyloidosis. It is a particular object of the invention to provide a method for diagnosing, monitoring, and treating neurodegenerative diseases associated with amyloidosis, in particular Alzheimer""s disease, by measuring and inhibiting the formation of AGE-xcex2AP. It is another particular object to provide a method for diagnosing, monitoring, and treating diabetes Type II by measuring and inhibiting the formation of AGE-amylin.
It is a still further object of the present invention to provide a method for identifying new drugs and corresponding agents capable of treating abnormal amyloid polypeptide aggregation, in one aspect by use of an assay involving AGE-amyloid polypeptide, in particular AGE-xcex2AP or AGE-amylin.
Still another object of the invention is to provide for removing amyloid plaques that have formed in a subject by activating the mechanisms for recognition and removal of AGE-amyloid in the body of a subject, and which may be directly or indirectly responsible for a pathology.
It is yet another object to utilize AGE-amyloid polypeptides, particularly AGE-xcex2AP and AGE-amylin, to treat systemic or neurodegenerative diseases associated with amyloidosis, in particular Alzheimer""s disease and Type II diabetes, respectively.
It is still a further object of the present invention to identify AGE-amyloid proteins and methods of inhibiting their formation in instances or disease conditions where the presence or biological activity of these AGE-amyloid proteins is detrimental to the host organism, or indicative of the presence of a disease state in the host organism.
Other objects and advantages will be apparent from a consideration of the ensuing detailed description which proceeds with reference to the following illustrative drawings.